There is a general need for readily regulatable promoters to control the amount and the timing of gene expression in a variety of experimental and commercial applications. In particular, homogeneous expression of a recombinant gene in all cells of the population is an important factor for successful protein production to perform metabolic optimization rather than massive overexpression. However, the available repertoire of Escherichia coli expression systems (lactose-inducible Plac and arabinose-inducible PBAD) are subject to all-or-none gene expression, in which intermediate inducer concentrations produce an inhomogeneous culture with a subpopulation of cells that is fully induced and a subpopulation that is uninduced (Novick 1957, Siegele 1997).
Recently, homogenous expression from PBAD was achieved (Khlebnikov 2000) by placing arabinose transporter gene under control of a promoter that was not regulated by arabinose. However, this system uses the inexpensive sugar (L-arabinose) as an inducer and is somewhat weaker than the Plac promoter (Baneyx 1999). In a similar vein, the all-or-none induction of lactose-inducible promoters can be eliminated through the use of a gratuitous inducer, IPTG, that readily diffuses across the cell wall (Khlebnikov 2002) and a lacY deletion mutant because the uptake of IPTG is largely mediated by the proton symport lac permease (Jensen 1993). Despite many merits and wide usage, the lactose-inducible promoter systems, together with T7-based expression system that is based on lactose promoters, require the expensive inducer IPTG as an inducer. For large-scale or repeat experiments such methods may become rather unattractive options (Su 1990). Also, IPTG could easily contaminate the protein products due to its indigestibility by the cells (Figge 1988). Moreover, the use of IPTG for production of human therapeutic proteins is undesirable because of its toxicity (Figge 1988).
T7-based expression systems (Studier 1990) are widely used for large scale over-expression of recombinant proteins in both bacterial (Tabor 1985, Studier 1986) and eukaryotic cells (Fuerst 1986, Dunn 1988). The system comprises T7 gene 1 encoding T7 RNA polymerase, which specifically interacts with the T7 promoter (Tabor 1985). In particular, T7 RNA polymerase exhibits superior processability as compared to E. coli housekeeping RNA polymerase (Studier 1986). Despite great successes in using the T7 expression system for protein production, it has considerable problems. To be used in E. coli, a bacterial strain was engineered to carry a chromosomal copy of T7 gene 1 under the control of lacUV5 promoter, and an expression vector containing the T7 promoter was constructed. The most commonly encountered problems of the T7 system are inconsistency in levels of expression, formation of inclusion bodies (Hoang 1999, Jeong 1999) and instability of clones (Hattman 1985). Inconsistency in levels of expression of target genes has been attributed to the leaky expression of T7 RNA polymerase, although the gene is placed under control of strong inducible promoters. Gene products that severely affect the host cell's growth rate at low concentrations are considered to be toxic and can make it difficult to stably maintain plasmids (Hattman 1985).
More recently, the thermoregulatable T7 expression system was reported for a simple and inexpensive way to operate (Chao 2002, Wang 2004). Also, cold-shock expression vectors were developed to substitute the widely used pET vectors (Qing 2004). However, these two systems have a disadvantage in that the gene fused to the T7 promoter on the plasmid can only be actively expressed at higher or lower temperature rather than optimal growth temperature.
There is a need in the art for expression vectors that avoid the above-mentioned drawbacks. The present invention addresses this need.